Project Summary/Abstract In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type zebrafish lines for a wide range of genetic and biomedical research. Maintaining all of these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Cryopreservation of zebrafish sperm, eggs and embryos is vital to the strategy of NIH?s Division of Comparative Medicine, which envisions increased multi-institutional research using animal models. To date, zebrafish sperm and eggs have been successfully cryopreserved, but zebrafish embryos have not. The main challenges with zebrafish embryo cryopreservation are the large size of the embryo, which limits the rate at which the embryo can be externally cooled and warmed, and a multi-compartmental embryo with different permeabilities preventing uniform diffusion of cryoprotectant agents. Methods and apparatus that enabled the long-term storage and transport of cryopreserved embryos would address a critical need for zebrafish researchers. This project will develop and optimize a gold nanoparticle (GNP) based rapid-warming technology that has successfully generated viable zebrafish grown from a cryopreserved embryos. Due to the large size of the embryo, traditional warming mechanisms are too slow and result in the formation of ice crystals that damage the embryos and prevent them from being viable. The key innovation of this project addresses this limitation in the warming step. Injected GNP act as a distributed network of ultra-efficient heaters that generate warming rates of millions of C/min when illuminated with an infrared laser. To achieve this ultrafast and reproducible warming, stable, low-toxicity GNPs with strong absorption at the laser wavelength will be fabricated and their photothermal properties measured. A micro-injection technique developed at the University of Minnesota will be optimized to circumvent the permeability barrier to safely introduce cryoprotectant agents (CPAs) and GNPs into the yolk and chorion compartments. Embryos will be rapidly cooled to prevent damaging ice formation, and laser warming techniques will be optimized to increase embryo survival after thawing. The combined foundational research of Dr. Bischof and his collaborators along with the expertise in GNP synthesis and manufacturing at nanoComposix will allow for rapid optimization and commercialization of this technology.